Aggregation and fibrillization of ?-synuclein has been implicated in the progression of Parkinson's Disease, which currently has no cure. This research project is focussed on the molecular steps for initiation of ?-synuclein aggregation and fibrillization. The project builds on the PI's discovery that ?-synuclein appears resistant to aggregation except when exposed to non-fluid hydrophobic interfaces or to preformed seeds. The research uses chemically functionalized silica substrates to measure ?-synuclein conformation at the interface, and uses identically functionalized mixing balls to assay for the interfacial influence on aggregation and fibrillization. This platform allows systematic evaluation of the relationship between the physical and chemical properties of the interface, the conformational changes in ?-synuclein, and the initiation of aggregation and fibrillization. The research will identify the physical and chemical properties of interface and the changes in ?-synuclein structure that lead to aggregation and fibrillization. The project then builds on the identification of that first step of aggregation and fibrillization to develop a fluorescence assayfor the changes in ?-synuclein that occur during the first step(s). The assay is intended to enable single-molecule sensitivity. The assay will be used to learn more about the first steps of ?-synuclein aggregation and fibrillization. To evaluate the hypothesis that ?-synuclein resists fibrillization without a nucleation partner, samples free from nucleating interfaces will be evaluated to try to find conditions for fibrillization that do not include nucleating parnters. Fially the fluorescence assays developed to probe the first steps of aggregation leading to fibrillization will be used to evaluate macromolecular binding partners. The macromolecules will be classified using the assay according to the type of structure they induce in ?-synuclein and how they inhibit or promote aggregation and fibrillization. These assays will evaluated for their future utiity as discovery reagents for ?-synuclein binding partners in cell lysates.